RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors

Gain-of-function mutations in the LRRK2 gene cause Parkinson’s disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adapter MADD, potentially preventing formation of the RAB3A-MADD-KIF1A/1Bβ complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD.


INTRODUCTION
Parkinson's disease (PD) is a devastating neurodegenerative disease that causes cardinal motor symptoms: rest tremor, rigidity, bradykinesia, and postural instability 1 . These are manifestations of the loss of select neuronal populations, most prominently dopaminergic neurons in the substantia nigra pars compacta (SNc). In addition to these motor symptoms, PD is clinically characterized by debilitating nonmotor symptoms such as cognitive decline, dementia, sleep disturbance, and depression 1 , suggesting that pathogenic mechanisms may also alter synaptic transmission in a broader set of neuronal populations.
Autosomal dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of PD, accounting for ~5% of familial cases 2 . Furthermore, genome-wide association studies have implicated LRRK2 noncoding variants in sporadic PD. Seven gain-of-function pathogenic mutations in LRRK2 increase LRRK2 kinase activity, resulting in elevated phosphorylation of a subset of RAB GTPases (RABs) 3 . RABs coordinate vesicle trafficking by selectively associating with membrane compartments and recruiting effector proteins 4 . Mounting evidence shows that LRRK2mediated phosphorylation of RABs alters their binding properties, either by introducing a new set of binding partners [5][6][7][8][9] or by impairing interaction with previous partners 3, 10,11 . Therefore, the relative activity of LRRK2 and its opposing protein phosphatase 1H (PPM1H) regulates RAB binding to effectors 12, 13 .
In recent work, we demonstrated that LRRK2-mediated RAB hyperphosphorylation has consequences for retrograde axonal transport of autophagic vesicles (AVs), disrupting an interplay of motor regulators in a manner scaling with magnitude of LRRK2 kinase activity 12 . Neurons require the directed transport of a wide range of distinct axonal cargoes to maintain homeostasis and synaptic function, in addition to AVs. Given the cognitive impairments and other non-motor manifestations of PD, an axonal cargo of particular interest is the synaptic vesicle precursor (SVP). SVPs arise in the neuronal soma and are transported anterogradely by kinesin-3 family members KIF1A and KIF1Bβ, carrying proteins that are fated for mature synaptic vesicles (SVs) at presynaptic sites [14][15][16][17][18][19][20] , including numerous en passant synapses populating the complex axonal arbor. These SV proteins are only recycled for a limited time before being
To test this hypothesis, we employed human induced pluripotent stem cells (iPSCs) with heterozygous KI of LRRK2-p. R1441H. These iPSCs had been gene-edited by the iPSC Neurodegenerative Disease Initiative (iNDI) at the NIH 34 to introduce the p.R1441H mutation at the endogenous LRRK2 locus of the KOLF2.1J parental line. Using tetracycline-inducible expression of NGN2, we differentiated these iPSCs into excitatory glutamatergic neurons (iNeurons) 35 . The resulting p.R1441H KI iNeurons exhibit elevated RAB phosphorylation, as previously shown using an antibody pan-specific to multiple phosphorylated RABs including RAB3A 12 . To assess SVP trafficking in these mutant iNeurons and corresponding WT control iNeurons, we live-imaged SVPs labeled by the fluorescent reporter mScarlet-synaptophysin (SYP) ( Figure 1A). We imaged each neuron at the proximal axon in order to limit variability caused by axonal branchpoints, at a distance approximately 100 μm from the soma in order to avoid the axonal initial segment. To more clearly visualize SYP+ vesicles entering the imaged axonal region, we photobleached the field of view prior to imaging to deplete pre-existing mScarlet-SYP signal in the axon ( Figure 1A).
In WT iNeurons we observed rapid, highly-processive SVPs traveling in the anterograde direction ( Figure 1B). To accommodate the high speed of these vesicles, we imaged each axon at 5 frames per second for 5 minutes. We observed the anterograde population of SYP+ vesicles to be more numerous, rapid, and processive than the retrograde population ( Figure 1B, inset), consistent with our previous observations in WT primary mouse hippocampal neurons 22 and in WT iNeurons from a different parental line 36 (Aiken and Holzbaur, 2023; manuscript in preparation). In LRRK2-p.R1441H KI iNeurons, we observed a significant decrease in anterograde SVP flux ( Figure 1B,C). However, there was no effect on the velocity of anterograde vesicles ( Figure 1D), indicating that SVPs that entered the axon were . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint transported normally. We did not observe a change in the flux of retrograde SYP+ vesicles ( Figure 1E), suggesting that expression of mutant LRRK2 specifically affected the anterograde population.
To confirm that this effect is dependent on LRRK2 kinase activity, we applied the selective LRRK2 kinase inhibitor MLi-2 37 to p.R1441H KI iNeurons ( Figure 1F). Overnight treatment with 100 nM MLi-2 rescued anterograde SVP flux ( Figure 1G,H) without affecting anterograde velocity ( Figure   1I). MLi-2 treatment had no effect on flux of retrograde SYP+ vesicles ( Figure 1J). In parallel experiments, we also examined SVP flux in i 3 Neurons 8,38,39 gene-edited from the WTC11 parental line to express the common pathological p.G2019S variant of LRRK2. This mutation also hyperactivates kinase activity but to a lesser extent than the p.R1441H mutation 3,12,31 . In p.G2019S KI i 3 Neurons, we found that overnight treatment with 100 nM MLi-2 increased anterograde SVP flux compared to DMSO treatment (Figure S1A-C), albeit to a lesser extent than was observed in iNeurons expressing p.R1441H.
Together, these results show a kinase activity-dependent decrease in anterograde axonal SVP flux caused by hyperactive LRRK2. Importantly, we did not observe altered anterograde SVP velocity, nor did we observe altered retrograde flux, suggesting that p.R1441H's effect is specific to decreasing the number of SVPs that enter the axon from the soma.

PPM1H KO phenocopies the effect of hyperactive LRRK2 on anterograde SVP flux in the axon
PPM1H is a phosphatase that opposes the activity of LRRK2 through dephosphorylation of RAB GTPases (Figure 2A) 13,40,41 . Therefore, the balance of LRRK2 and PPM1H activity has the potential to regulate neuronal pathways, including transport of axonal cargoes, by modulating RAB phosphorylation levels 12 . We previously generated PPM1H KO iPSCs from the same KOLF2.1J parental line as the p.R1441H KI iPSCs used here 12 . To determine whether loss of PPM1H would phenocopy the effect of hyperactive LRRK2-p.R1441H on anterograde SVP flux, we compared mScarlet-SYP motility in WT and PPM1H KO iNeurons ( Figure 2B). Indeed, we observed decreased anterograde axonal flux of the SYP+ population upon loss of PPM1H ( Figure 2C,D). Similar to observations in p.R1441H KI neurons, there was no change in anterograde velocity in PPM1H KO cells ( Figure 2E). While we did observe a significant decrease in retrograde SYP+ vesicles in PPM1H KO iNeurons ( Figure 2F), the size of the effect was much smaller than the effect on the anterograde population ( Figure 2D).
In sum, these results from orthogonal models indicate that either hyperactive LRRK2 activity . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

RAB hyperphosphorylation alters compartmental distribution of SVP-associated proteins
SVP transport has the important role of replenishing presynaptic sites with synaptic vesicle (SV) proteins 21,22 . We next asked whether decreased anterograde transport of SVPs caused by RAB hyperphosphorylation has consequences on distribution of synaptic proteins within the neuron.
Specifically, we interrogated the localization of two SV proteins known to be trafficked with SVPs and delivered to presynaptic sites: SYP and synaptobrevin-2 (SYB2).
To examine somal content, we stained endogenous SYP and SYB2 in p.R1441H KI and PPM1H KO iNeurons, as well as control WT KOLF2.1J iNeurons. An antibody to endogenous microtubuleassociated protein-2 (MAP2) signal was used to visualize the somatodendritic compartment ( Figure 3A).
In both RAB-hyperphosphorylated conditions, we detected significant increases in the somal intensity of SYP ( Figure 3A Proteins fated for SVPs are believed to be sorted at the trans-Golgi network (TGN) [42][43][44] . We next explored whether a portion of somal SYP may be sequestered at the TGN in the context of elevated RAB3A phosphorylation. WT, p.R1441H KI, and PPM1H KO iNeurons were stained for endogenous SYP and golgin-97, a TGN marker ( Figure S2E). Again, MAP2 signal was used to visualize the somatodendritric compartment. In all three conditions, we observed that SYP intensity is enriched at the TGN relative to whole soma ( Figure S2F). Compared to WT neurons, PPM1H KO neurons displayed increased SYP intensity that co-localized with golgin-97 ( Figure S2F). However, we did not detect this effect in p.R1441H KI iNeurons ( Figure S2F).
Next, we sought to determine whether hyperphosphorylation of RABs disrupts delivery of synaptic proteins to presynaptic sites. To accomplish this, we employed a recently developed heterologous synapse model for human neurons that allows unambiguous analysis of trafficking to the presynaptic compartment 36 (Aiken and Holzbaur, 2023; manuscript in preparation), introducing nonneuronal human embryonic kidney (HEK) 293 cells expressing the postsynaptic ligand neuroligin-1 (NL1) into co-culture with iNeurons ( Figure 3D). Within 24 hours of introducing HEK cells, iNeuron axons specifically recognize NL1-expressing HEK cells and form connections where presynaptic proteins accumulate ( Figure 3D, inset). This system provides both spatial and temporal control for quantification of SVP-associated protein accumulation. These heterologous presynapses contain SYP, SYB2, synapsin I/II (SYN), VGLUT1, and SVs that cycle upon neuronal depolarization 36 (Aiken and Holzbaur, 2023; manuscript in preparation). In heterologous synaptic cultures stained for endogenous SYP and SYB2 ( Figure 3E), quantification revealed significantly decreased accumulation of both SYP ( Figure 3F) and SYB2 ( Figure 3G) at presynapses in PPM1H KO iNeurons relative to WT. In p.R1441H KI iNeurons, . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint SYP presynaptic accumulation was also decreased, with SYB2 presynaptic accumulation trending downward but not achieving statistical significance ( Figure 3F-G).
Together, these experiments show that the compartmental distribution of two different SVPassociated proteins is disrupted in p.R1441H KI and PPM1H KO iNeurons, with increased somal abundance and decreased presynaptic content. Together with our live-imaging findings, these data are consistent with somal sequestration and impaired presynaptic delivery of SVP-associated proteins as a consequence of disrupted anterograde SVP transport.

Phosphorylation of RAB3A impairs interaction with motor adaptor protein MADD
MADD has been previously shown to be essential for transport of RAB3-containing SVPs by KIF1A and KIF1Bβ 16 . The same study showed that MADD directly interacts with KIF1A/1Bβ and RAB3. A more recent study further demonstrated that MADD interacts selectively with SVPs, not other axonal cargoes including dense core vesicles (DCVs) and lysosomes 18 . MADD is also known as RAB3-GEP (guanine nucleotide exchange protein) due to its role as a GDP-GTP exchange factor (GEF) for RAB3 23 . Notably, anterograde SVP transport has been shown to depend on the GTP-bound state of RAB3 16,24 . To probe the mechanism underlying LRRK2-p.R1441H's effect on anterograde axonal SVP flux, we first tested whether overexpressing the GTP-locked mutant of RAB3 would rescue the deficit. Of the four RAB3 isoforms, we chose to focus on the best-characterized isoform, RAB3A, which is also the most abundant in the cortex 25,26 . We found that transient expression of the glutamine-to-leucine (Q81L) mutant RAB3A, predicted to lock RAB3A into a GTP-bound state ( Figure 4A It has previously been shown that LRRK2-mediated phosphorylation of RAB8A disrupts its ability to bind to RABIN8, its cognate GEF 3,11 . We therefore investigated whether the known interaction between RAB3A and MADD is altered by RAB3A phosphorylation. To test this, we co-expressed HA-MADD in HEK293 cells with EGFP-labeled RAB3A, with or without point mutations at the threonine residue that is phosphorylated by LRRK2 ( Figure 4A) 40,41 . Consistent with previous work 16 , RAB3A WT co-immunoprecipitated with MADD ( Figure 4E). Threonine-to-alanine (T86A) mutant RAB3A, predicted to be non-phosphorylatable, exhibited the highest binding affinity to MADD ( Figure 4E,F). Threonine-toglutamic acid (T86E) mutant RAB3A, predicted to be a phosphomimetic, bound more weakly to MADD than RAB3A WT ( Figure 4E,F).
Compared to the T86A and T86E mutants, RAB3A WT exhibited an intermediate binding affinity with MADD ( Figure 4E,F). This raised the intriguing possibility that a fraction of the transientlyexpressed EGFP-RAB3A WT was phosphorylated by HEK cell endogenous LRRK2 WT and thus exhibited . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. an impaired ability to bind MADD. To confirm that phosphorylation of RAB3A WT affects MADD binding, we applied lambda protein phosphatase (λPP) to lysates prior to co-immunoprecipitation of EGFP-RAB3A WT and HA-MADD ( Figure 4G). λPP treatment effectively decreased levels of phosphorylated EGFP-RAB3A WT in the bound fraction ( Figure 4H) and increased binding to HA-MADD ( Figure 4G,I).
In sum, our results show that interaction between RAB3A and MADD is impaired by RAB3A phosphorylation at the T86 residue. Given MADD's dual role as a GEF for RAB3A and a motor adaptor for RAB3A-positive SVPs, these data suggest that impaired RAB3A-MADD interaction contributes to the deficit of anterograde SVP flux in p.R1441H KI iNeurons ( Figure 1A-C), which can be rescued by overexpression of GTP-locked RAB3A ( Figure 4D).

Phosphorylation of RAB3A impairs interactions with RAB-GDI1 and RAB3GAP but not RIM2 or synapsin
Multiple regulatory proteins determine RAB GTPase localization and binding state 4 . GEFs such as MADD recruit and drive conversion of RABs to the active GTP-bound state at membranes 23 . Each RAB functions at specific membranes, contributing to membrane identity by selective effector recruitment 45 . However, cytosolic GDP-bound RABs have been shown to be "promiscuous" in terms of their ability to enter membranes belonging to a wide range of intracellular organelles, where they may fail to encounter their cognate GEF 46 . Two regulatory proteins called RAB3 GTPase-activating protein (RAB3GAP) and RAB GDP dissociation inhibitor-1 (RAB-GDI1) act in concert to retrieve RABs from inappropriate membranes. RAB3GAP converts GTP-bound RABs to the GDP-bound state, and RAB-GDI1 serves as a chaperone to return GDP-bound RABs from membranes to the cytosol 4 . Dysregulation of RAB GTP binding state may therefore alter subcellular RAB localization and contribute to reduced effective availability of RABs 4,47 .
Previous work showed that phosphomimetic mutant RAB GTPases fail to bind RAB-GDI1, and that this is also true for directly-phosphorylated RAB8A WT and RAB12 WT 3,10,48 . Consistent with these findings, in co-immunoprecipitation experiments we observed that the phosphomimetic T86E mutation abolished the interaction between EGFP-RAB3A and endogenous RAB-GDI1, compared to the non- confirming that direct RAB3A phosphorylation disrupts the interaction between RAB3A and RAB-GDI1.
In the same set of co-immunoprecipitation experiments, we also explored whether phosphorylation of RAB3A disrupts binding to RAB3GAP, quantified with an antibody for the non-. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint catalytic subunit RAB3GAP2. We noted greater non-specific binding of RAB3GAP than GDI1 to the EGFP vector ( Figure 5A), which was subtracted prior to quantification. We observed that binding of EGFP-RAB3A to RAB3GAP was decreased by the phosphomimetic T86E mutation compared to both RAB3A WT and RAB3A TA ( Figure 5A,D). Interestingly, while the T86E mutation strongly disrupted the RAB3A-GDI1 interaction ( Figure 5A,C), this mutation had a more moderate effect on the RAB3A-RAB3GAP interaction ( Figure 5A,D).
RAB3A has been implicated in mechanisms of SV release, acting in concert with effector proteins [49][50][51] . Given the effect of RAB3A phosphorylation on binding to MADD, RAB-GDI1, and RAB3GAP, we wondered if RAB3A phosphorylation indiscriminately impaired interaction with all of its effectors. We therefore tested phosphomimetic mutant RAB3A binding to RAB3A-interacting molecule 2 (RIM2) and synapsin, two presynaptic proteins that have been shown to act as RAB3A effectors 50,52 . In agreement with these previous reports, we observed pulldown of both RIM2 ( Figure S3A) and synapsin ( Figure S3B) by RAB3A WT . However, neither the phosphomimetic T86E mutation nor the nonphosphorylatable T86A mutation affected RAB3A binding to either RIM2 or synapsin, in contrast to MADD, GDI, and RAB3GAP.
In summary, our results show that RAB3A phosphorylation at T86 selectively regulates binding to a subset of partners. Binding to the regulatory chaperone protein RAB-GDI1 was strongly impaired, while moderate disruption was observed for binding to the motor adapter MADD and the regulatory protein RAB3GAP, and no effects were observed on binding to either RIM2 or synapsin.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. Though the LRRK2 substrate RAB3A has long been known to be essential for axonal SVP transport, the consequences of RAB3A phosphorylation on this trafficking pathway have not previously been explored. Here, we show that in two iNeuron models of RAB hyperphosphorylation (LRRK2-p.R1441H KI and PPM1H KO), we observe impaired anterograde flux of SVPs (Figure 1,2). We find that RAB3A phosphorylation at T86 disrupts binding to the motor adapter protein MADD (Figure 4). Phosphorylation of RAB3A also alters binding affinity to the regulatory proteins RAB3GAP and RAB-GDI1 ( Figure 5).
Our findings support a model where pathogenic hyperactive LRRK2 causes dysregulated binding of RAB3A in the neuronal soma, including impairment of the formation of the RAB3A-MADD-KIF1A/Bβ complex ( Figure 6). We hypothesize that this contributes to the reduced availability of RAB3 to stimulate anterograde SVP transport. Consistent with this hypothesis, we find that the compartmental distribution of SVP-associated proteins is disrupted within neurons with hyperphosphorylated RABs, manifesting as increased somal content and decreased delivery to presynaptic sites along the axon (Figure 3).
Our previous work linked hyperactive LRRK2 mutations to the disruption of the retrograde axonal transport of AVs 8,12 , most likely mediated by RAB10 and/or RAB35 5,6,13,53 . In contrast, hyperactive LRRK2 does not alter the axonal transport of mitochondria 12 , consistent with our current understanding that there is no known role for RABs in regulating the axonal transport of these organelles. Given the altered transport of RAB3A+ SVPs, our findings indicate a high degree of RAB-dependent selectivity for which axonal cargoes are perturbed by pathogenic hyperactive LRRK2. Furthermore, because PPM1H KO phenocopies these transport defects, this implies that the balance between LRRK2 WT and PPM1H may regulate transport of both SVPs and autophagosomes under physiologic conditions. As at least ten different RABs are endogenously phosphorylated by LRRK2 10 , it remains to be explored whether other axonal cargoes rely on RAB-mediated transport mechanisms that are regulated by LRRK2.
Recent work indicates that the subcellular co-localization of PPM1H with specific RABs strongly influences levels of RAB phosphorylation 54 . Regulation of RAB-mediated pathways in neurons are therefore determined by the balance of LRRK2 and PPM1H activities at each specific membrane compartment, in ways that are difficult to predict from whole-cell pRAB levels alone. While our results show that both p.R1441H KI and PPM1H KO affect SVP transport and synaptic protein distribution, the effect size was generally more pronounced in PPM1H KO neurons ( Figure 3F,G; Figure S2F). Notably, PPM1H has been shown to strongly localize to the Golgi 13,54 , suggesting that its loss may cause more striking effects on protein sorting and cargo loading at the TGN. Further work could reveal how relative . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made    Figure S4A) 16 . Moreover, MADD's motor-binding death domain region is found toward its C-terminus 16,18 . RABs are phosphorylated by LRRK2 at their characteristic switch II domain, which changes conformation in response to nucleotide binding in order to allow for interaction with effectors or regulatory proteins 3,58 . Thus, phosphorylation of RAB3A likely disrupts interaction between the switch II region of RAB3A and the N-terminus of MADD, without interrupting MADD's ability to bind kinesin-3 ( Figure S4A). Further work is required to elucidate the order of events and kinetics by which pRAB3A interrupts loading of SVP cargo onto the MADD-kinesin motor complex. However, we observed that overexpression of GTP-locked RAB3A rescued anterograde SVP flux in p.R1441H KI iNeurons ( Figure 4B-D), suggesting that increasing levels of active RAB3A in this system is sufficient to restore appropriate levels of the RAB3A-MADD-KIF1A/Bβ complex. This indicates that the increased fraction of phosphorylated RAB3A induced by p.R1441H KI reduces the abundance of eligible RAB3A required for the initiation of SVP transport.
Here, we primarily focused on the transport dynamics of the anterograde population of SYP+ vesicles, for which the mechanism of rapid, highly processive transport is known 22 . These results demonstrate that the major effect of RAB3A phosphorylation is on flux of the anterograde SYP+ population. The transport dynamics of the retrograde SYP+ population are not as well-characterized. Across multiple studies in mammalian neurons, we have observed it to be more heterogeneous, and overall less numerous, rapid, and processive than the anterograde population 22,36 (Aiken and Holzbaur, 2023; manuscript in preparation). Recent work from our group has identified that SV proteins (including SYP and . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. on SV exocytosis. LRRK2 has been reported to interact with or regulate actin, synapsin I, SNAP25, syntaxin, NSF, endophilin A, dynamin, auxillin, and synaptojanin, all of which contribute to the SV cycle [59][60][61][62][63][64][65][66][67][68] . Furthermore, LRRK2 substrates RAB3A 49-51,69,70 , RAB5 [71][72][73] , and RAB35 74,75 have been implicated in membrane trafficking within the presynapse. The relative balance of LRRK2 and PPM1H activity at the presynapse will likely regulate some of these interactions, but others may involve scaffolding domains of LRRK2 that are not believed to be directly kinase-dependent 63,76 . RAB-mediated pathways at the presynapse may therefore be good candidates to be differentially affected by hyperactive LRRK2.
Ultimately, the effect of pathogenic LRRK2 mutations on synaptic transmission is likely an integrated function of these different interactions, with more work needed to uncover how these intersecting pathways may contribute to development of non-motor symptoms in PD.
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DATA AND RESOURCE AVAILABILITY Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Erika L. F. Holzbaur (holzbaur@pennmedicine.edu).

Materials Availability
Unique reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement. Plasmids generated in this study have been deposited to Addgene (identifier numbers listed in resources table).

Data Availability
• Primary data that is presented in this study has been deposited in Zenodo and repository and are publicly available as of the date of publication. These can be accessed using the Digital Object Identifier 10.5281/zenodo.8156734.
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DECLARATION OF INTERESTS
The authors declare no competing interests.
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Plasmids and reagents
Plasmids and reagents used are detailed in Table 1, along with Addgene identification numbers. Antibodies used are detailed in Table 2, along with application and dilution. CMV HA-RIM2 was derived from GST-RIM2-RBD which was a gift from Ruben Bierings (Erasmus University Medical Center). This construct contains the first 411 amino acids of RIM2, which contains the RAB binding domain 77 .

Piggybac-mediated iPSC-derived neuron differentiation
KOLF2.1J-background WT and LRRK2-p.R1441H KI iPSCs were a gift from B. Skarnes (Jackson Laboratories, Connecticut) as part of the iPSC Neurodegenerative Disease Initiative (iNDI) and have been described previously 35 . KOLF2.1J-background PPM1H KO iPSCs were generated as described previously 12 . Cytogenetic analysis of G-banded metaphases cells showed a normal male karyotype (Cell Line Genetics). Mycoplasma testing was negative. iPSCs were cultured on plates coated with Growth Factor Reduced Matrigel (Corning) and fed daily with Essential 8 media (Thermo Fisher). To stably express doxycycline-inducible hNGN2 using a PiggyBac delivery system, iPSCs were transfected with PB-TO-hNGN2 vector (gift from M. Ward, NIH, Maryland) in a 1:2 ratio (transposase:vector) using Lipofectamine Stem (Thermo Fisher). After 72 hours, transfected iPSCs were selected for 48 hours with 0.5 μg/mL puromycin (Takara). Differentiation of iPSCs into iNeurons was performed using an established protocol 35,38 . In brief, iPSCs were passaged using Accutase (Sigma) and plated on Matrigel-coated dishes was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint Pre-i 3 Neuron iPSCs (human iPSCs with an integrated doxycycline-inducible mNGN2 transgene in the AAVS1 safe-harbor locus) were a gift from M. Ward (National Institutes of Health, Maryland) and have been described previously 8,12,38,39 . Cytogenetic analysis of G-banded metaphases cells showed a normal male karyotype (Cell Line Genetics). Mycoplasma testing was negative. Pre-i 3 N iPSCs were cultured on plates coated with Growth Factor Reduced Matrigel (Corning) and fed daily with Essential 8 media (Thermo Fisher). Induction into neuronal fate with doxycycline and cryopreservation of pre-differentiated neurons was performed as described above ("Piggybac-mediated iPSC-derived neuron differentiation").

Live-cell imaging and motility quantification
iNeurons were imaged on DIV21 in low fluorescence Hibernate A medium (Brain Bits) supplemented with 2% B27, 10 ng/mL BDNF and 10 ng/mL NT-3. Neurons were imaged in an environmental chamber at 37°C. Recordings of mScarlet-SYP+ vesicles for Figures 1 and 4 were acquired on a PerkinElmer UltraView Vox Spinning Disk Confocal system with a Nikon Eclipse Ti inverted microscope, using a Plan Apochromat 60x 1.40 NA oil immersion objective and a Hamamatsu EMCCD C9100-50 camera controlled by Volocity software. Following a scheduled microscope upgrade, live imaging for Figure 2 and Figure 4 was instead performed using a Hamamatsu ORCA-Fusion C14440-20UP camera controlled by VisiView software. Axons were identified based on morphological parameters 39,80 , and measurements were made to image ~100-150 µm from the neuronal soma. After identifying this region, . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Immunostaining and quantification
At DIV14, human iNeurons were fixed in 4% paraformaldehyde supplemented with 4% sucrose for 15 minutes, washed four times with PBS, and permeabilized with 0.2% Triton-X in PBS for 15 min.
Cells were then blocked for 1 hour with 5% goat serum and 1% BSA in PBS. Neurons were then incubated in primary antibody (see Table 2) diluted in blocking solution at room temperature for 1 hour, washed three times with PBS, and incubated in secondary antibody (see Table 2 For experiments shown in Figure 3A-C, the MAP2 channel was used to select an ROI around the somatic compartment by a blinded investigator. This ROI was used to measure the mean grey value of the SYP and SYB2 signals using sum projections. SYP/SYB2 intensity for each neuron was normalized to the average intensity of the WT neurons from that experimental replicate. Figure  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07. 25.550521 doi: bioRxiv preprint For experiments shown in Figure 3D-G, HEK cells transfected 24 hours prior with 6 µL:1 µg mix of FUGENE and pBI-NL1-BFP (bicistronic vector expressing untagged NL1 and cytosolic BFP) were added to DIV13 iNeurons (100K transfected HEK cells added to DIV13 iNeurons cultured in 35 mm imaging dishes). 24 hours after addition of transfected HEK cells, at iNeuron DIV14, cells were fixed for immunocytochemistry in 4% paraformaldehyde supplemented with 4% sucrose and stained as described above (see Table 1 and 2 for antibody information). For analysis, the NL1-BFP channel was used to select an ROI around a NL1+ HEK cell. To determine SYP and SYB2 intensity within presynaptic regions spanning the NL1+ HEK ROI, max-projection images were created and FIJI's Thresholding tool was used to segment an 8-bit object mask based on the top 1% intensity of the SYP or SYB2 channel. FIJI's Analyze Particles tool was then used on the object mask redirected to the original SYP or SYB2 image channel to determine intensity values for individual presynaptic puncta. Figure  For experiments shown in Figure S1E-F, the MAP2 channel was used to select an ROI around the somatic compartment by a blinded investigator. This ROI was used to measure the mean grey value of the somatic SYP signal using a sum projection. The "Adjust Threshold" function in ImageJ was used to create a mask on the region of high-intensity golgin-97 signal, and this ROI was used to measure the mean grey value of the SYP signal co-localized with the TGN. Figure legends contain the statistical test used and specific p values for each quantification. RStudio version 2021.9.2.382 was used to perform a linear mixed effects model (LME; R package "nlme"). The genotype was treated as the fixed effect. The independent experiment/culture was treated as the random effects. For all quantifications, at least three independent experiments were analyzed.
Proteins were transferred to Immobilon-FL PVDF membranes (Millipore) using a wet blot transfer system. Membranes were then stained for total protein using LI-COR Revert 700 Total Protein Stain.
Following imaging of total protein stain, membranes were de-stained and blocked for 5 minutes with Bio-. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
Cells were maintained at 37°C in a 5% CO 2 incubator. Cells were tested for mycoplasma contamination routinely, using MycoAlert detection kit (Lonza, LT07). For co-immunoprecipitation experiments, cells were plated on three 10 cm tissue culture dishes per condition and transfected 24 h before lysis using FuGENE 6 (6-12 μg total plasmid DNA; Promega). Published protocol can be found on Protocols.io (dx.doi.org/10.17504/protocols.io.kxygx3zeog8j/v1).  were added to clarified lysate for a final reaction volume of 600 μL and incubated at 30°C for 30 minutes.

Co-immunoprecipitation experiments and quantification
The same was performed for λPP-negative conditions, with 24 μL ddH2O instead. After incubation with . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint λPP, incubation with beads was performed as described above. Published protocol can be found on Protocols.io (dx.doi.org/10.17504/protocols.io.6qpvr36o2vmk/v1).
Co-immunoprecipitation was analyzed by Western blot (see "Immunoblotting"). Proteins from the same experiment were processed in parallel and resolved on different acrylamide gels based on protein size: 6% for HA-MADD, 10% for RAB3GAP2, RAB-GDI1, and SNAP-Synapsin, and 12% for EGFP-RAB3A, pT RAB, EGFP vector, and HA-RIM2. Figure legends contain the statistical test used and specific p values for each quantification.

SUPPLEMENTAL MATERIAL
Supplemental data and legends relating to Figures 1, 3, and 5 can be found in Figures S1-S3. Figure   S4 includes AlphaFold and AlphaFold-Multimer predictions relating to Figures 4 and 5.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint Figure S3 A was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint

RAB3A
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. residues that were previously shown 16 to be necessary and sufficient for binding to RAB3. Right, annotated in magenta: the death domain toward the C-terminus of MADD that has been shown to be the motor-binding region 16,18 (B) AlphaFold predictions 40,41 of RAB3A and RAB-GDI1. (C) AlphaFold-Multimer 40,41,[55][56][57] prediction of complex formed by RAB3A and RAB3GAP1, the catalytic subunit of RAB3GAP. ipTM + pTM score for this prediction: 0.79404.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2023. ; https://doi.org/10.1101/2023.07.25.550521 doi: bioRxiv preprint